Dual-wavelength photo-killing of methicillin-resistant Staphylococcus aureus.

With the effectiveness of antimicrobials declining as antimicrobial resistance continues to threaten public health, we must look to alternative strategies for the treatment of infections. In this study, we investigated an innovative, drug-free, dual-wavelength irradiation approach that combines 2 wavelengths of light, 460 nm and 405 nm, against methicillin-resistant Staphylococcus aureus (MRSA). MRSA was initially irradiated with 460-nm light (90-360 J/cm2) and subsequently irradiated with aliquots of 405-nm light (54-324 J/cm2). For in vivo studies, mouse skin was abraded and infected with approximately 107 CFUs of MRSA and incubated for 3 hours before irradiating with 460 nm (360 J/cm2) and 405 nm (342 J/cm2). Naive mouse skin was also irradiated to investigate apoptosis. We found that staphyloxanthin, the carotenoid pigment in MRSA cells, promoted resistance to the antimicrobial effects of 405-nm light. In addition, we found that the photolytic effect of 460-nm light on staphyloxanthin attenuated resistance of MRSA to 405-nm light killing. Irradiation of 460 nm alone did not elicit any antimicrobial effect on MRSA. In a proof-of-principle mouse skin abrasion infection model, we observed significant killing of MRSA using the dual-wavelength irradiation approach. However, when either wavelength of light was administered alone, no significant decrease in bacterial viability was observed. Moreover, exposure of the dual-wavelength irradiation to naive mouse skin did not result in any visible apoptosis. In conclusion, a dual-wavelength irradiation strategy may offer an innovative, effective, and safe approach for the treatment of skin infections caused by MRSA.

Photoinactivation of Moraxella catarrhalis Using 405-nm Blue Light: Implications for the Treatment of Otitis Media.

Moraxella catarrhalis is one of the major otopathogens of otitis media (OM) in childhood. M. catarrhalis tends to form biofilm, which contributes to the chronicity and recurrence of infections, as well as resistance to antibiotic treatment. In this study, we aimed to investigate the effectiveness of antimicrobial blue light (aBL; 405 nm), an innovative nonpharmacological approach, for the inactivation of M. catarrhalis OM. M. catarrhalis either in planktonic suspensions or 24-h old biofilms were exposed to aBL at the irradiance of 60 mW cm-2 . Under an aBL exposure of 216 J cm-2 , a >4-log10 colony-forming units (CFU) reduction in planktonic suspensions and a >3-log10 CFU reduction in biofilms were observed. Both transmission electron microscopy and scanning electron microscopy revealed aBL-induced morphological damage in M. catarrhalis. Ultraperformance liquid chromatography results indicated that protoporphyrin IX and coproporphyrin were the two most abundant species of endogenous photosensitizing porphyrins. No statistically significant reduction in the viability of HaCaT cells was observed after an aBL exposure of up to 216 J cm-2 . Collectively, our results suggest that aBL is potentially an effective and safe alternative therapy for OM caused by M. catarrhalis. Further in vivo studies are warranted before this optical approach can be moved to the clinics.

Antimicrobial Blue Light Inactivation of Microbial Isolates in Biofilms.

BACKGROUND AND OBJECTIVES: Biofilms cause more than 80% of infections in humans, including more than 90% of all chronic wound infections and are extremely resistant to antimicrobials and the immune system. The situation is exacerbated by the fast spreading of antimicrobial resistance, which has become one of the biggest threats to current public health. There is consequently a critical need for the development of alternative therapeutics. Antimicrobial blue light (aBL) is a light-based approach that exhibits intrinsic antimicrobial effect without the involvement of exogenous photosensitizers. In this study, we investigated the antimicrobial effect of this non-antibiotic approach against biofilms formed by microbial isolates of multidrug-resistant bacteria. STUDY DESIGN/MATERIALS AND METHODS: Microbial isolates of Acinetobacter baumannii, Candida albicans, Escherichia coli, Enterococcus faecalis, MRSA, Neisseria gonorrhoeae, Pseudomonas aeruginosa, and Proteus mirabilis were studied. Biofilms were grown in microtiter plates for 24 or 48 hours or in the CDC biofilm reactor for 48 hours and exposed to aBL at 405 nm (60 mW/cm2 , 60 or 30 minutes). The anti-biofilm activity of aBL was measured by viable counts. RESULTS: The biofilms of A. baumannii, N. gonorrhoeae, and P. aeruginosa were the most susceptible to aBL with between 4 and 8 log10 inactivation after 108 J/cm2 (60 mW/cm2 , 30 minutes) or 216 J/cm2 (60 mW/cm2 , 60 minutes) aBL were delivered in the microplates. On the contrary, the biofilms of C. albicans, E. coli, E. faecalis, and P. mirabilis were the least susceptible to aBL inactivation (-0.30, -0.24, -0.84, and -0.68 log10 inactivation, respectively). The same aBL treatment in biofilms developed in the CDC biofilm reactor, caused -1.68 log10 inactivation in A. baumannii and -1.74 and -1.65 log10 inactivation in two different strains of P. aeruginosa. CONCLUSIONS: aBL exhibits potential against pathogenic microorganisms and could help with the significant need for new antimicrobials in clinical practice to manage multidrug-resistant infections. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Quinine Improves the Fungicidal Effects of Antimicrobial Blue Light: Implications for the Treatment of Cutaneous Candidiasis.

BACKGROUND AND OBJECTIVE: Candida albicans is an opportunistic fungal pathogen of clinical importance and is the primary cause of fungal-associated wound infections, sepsis, or pneumonia in immunocompromised individuals. With the rise in antimicrobial resistance, it is becoming increasingly difficult to successfully treat fungal infections using traditional antifungals, signifying that alternative non-traditional approaches must be explored for their efficacy. STUDY DESIGN/MATERIALS AND METHODS: We investigated the combination of antimicrobial blue light (aBL) and quinine hydrochloride (Q-HCL) for improved inactivation of C. albicans, in vitro and in vivo, relative to either monotherapy. In addition, we evaluated the safety of this combination therapy in vivo using the TUNEL assay. RESULTS: The combination of aBL (108 J/cm2 ) with Q-HCL (1 mg/mL) resulted in a significant improvement in the inactivation of C. albicans planktonic cells in vitro, where a 7.04 log10 colony forming units (CFU) reduction was achieved, compared with aBL alone that only inactivated 3.06 log10 CFU (P < 0.001) or Q-HCL alone which did not result in a loss of viability. aBL + Q-HCL was also effective at inactivating 48-hour biofilms, with an inactivation 1.73 log10 CFU at the dose of 108 J/cm2 aBL and 1 mg/mL Q-HCL, compared with only a 0.73 or 0.66 log10 CFU by aBL and Q-HCL alone, respectively (P < 0.001). Transmission electron microscopy revealed that aBL + Q-HCL induced morphological and ultrastructural changes consistent with cell wall and cytoplasmic damage. In addition, aBL + Q-HCL was effective at eliminating C. albicans within mouse abrasion wounds, with a 2.47 log10 relative luminescence unit (RLU) reduction at the dose of 324 J/cm2 aBL and 0.4 mg/cm2 Q-HCL, compared with a 1.44 log10 RLU reduction by aBL alone. Q-HCL or nystatin alone did not significantly reduce the RLU. The TUNEL assay revealed some apoptotic cells before and 24 hours following treatment with aBL + Q-HCL. CONCLUSION: The combination of aBL + Q-HCL was effective at eliminating C. albicans both in vitro and in vivo. A comprehensive assessment of toxicity (cytotoxicity and genotoxicity) is required to fully determine the safety of aBL + Q-HCL therapy at different doses. In conclusion, the combination of aBL and Q-HCL may be a viable option for the treatment of cutaneous candidiasis. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Antimicrobial Blue Light Inactivation of Polymicrobial Biofilms.

Polymicrobial biofilms, in which mixed microbial species are present, play a significant role in persistent infections. Furthermore, polymicrobial biofilms promote antibiotic resistance by allowing interspecies transfer of antibiotic resistance genes. In the present study, we investigated the effectiveness of antimicrobial blue light (aBL; 405 nm), an innovative non-antibiotic approach, for the inactivation of polymicrobial biofilms. Dual-species biofilms with Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) as well as with P. aeruginosa and Candida albicans were reproducibly grown in 96-well microtiter plates or in the CDC biofilm reactor for 24 or 48 h. The effectiveness of aBL inactivation of polymicrobial biofilms was determined through colony forming assay and compared with that of monomicrobial biofilms of each species. aBL-induced morphological changes of biofilms were analyzed with confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). For 24-h old monomicrobial biofilms formed in 96-well microtiter plates, 6.30-log10 CFU inactivation of P. aeruginosa, 2.33-log10 CFU inactivation of C. albicans and 3.48-log10 CFU inactivation of MRSA were observed after an aBL exposure of 500 J/cm2. Under the same aBL exposure, 6.34-log10 CFU inactivation of P. aeruginosa and 3.11-log10 CFU inactivation of C. albicans were observed, respectively, in dual-species biofilms. In addition, 2.37- and 3.40-log10 CFU inactivation were obtained in MRSA and P. aeruginosa, dual-species biofilms. The same aBL treatment of the biofilms developed in the CDC-biofilm reactor for 48 h significantly decreased the viability of P. aeruginosa monomicrobial and polymicrobial biofilm when cocultured with MRSA (3.70- and 3.56-log10 CFU inactivation, respectively). 2.58-log10 CFU inactivation and 0.86-log10 CFU inactivation was detected in MRSA monomicrobial and polymicrobial biofilm when cocultured with P. aeruginosa. These findings were further supported by the CLSM and SEM experiments. Phototoxicity studies revealed a no statistically significant loss of viability in human keratinocytes after an exposure to 216 J/cm2 and a statistically significant loss of viability after 500 J/cm2. aBL is potentially an alternative treatment against polymicrobial biofilm-related infections. Future studies will aim to improve the efficacy of aBL and to investigate aBL treatment of polymicrobial biofilm-related infections in vivo.

Evaluating the Potential for Resistance Development to Antimicrobial Blue Light (at 405 nm) in Gram-Negative Bacteria: In vitro and in vivo Studies.

Antimicrobial resistance is a threat to public health that requires our immediate attention. With increasing numbers of microbes that are becoming resistant to routinely used antimicrobials, it is vital that we look to other, non-traditional therapies for the treatment of infections. Antimicrobial blue light (aBL) is an innovative approach that has demonstrated efficacy for the inactivation of an array of microbial pathogens. In the present study, we investigated the potential for resistance development to aBL in Gram-negative pathogenic bacteria by carrying out multiple aBL exposures on bacteria. In the first aBL exposure, clinical isolates of Pseudomonas aeruginosa, Acinetobacter baumannii, and uropathogenic Escherichia coli [107 colony forming units/mL (CFU/mL)] were irradiated in phosphate-buffered saline with aBL at 405 nm until a >99.99% reduction in bacterial viability was achieved. Irradiation was then repeated for each bacterial species over 20 cycles of aBL exposure. The potential for resistance development to aBL was also investigated in vivo, in superficial mouse wounds infected with a bioluminescent strain of P. aeruginosa (PAO1; 108 CFU) and irradiated with a sub-curative radiant exposures of 108 or 216 J/cm2 aBL over 5 cycles of treatment (over 5 days) prior to bacterial isolation from the animal tissue. PAO1 isolated from infected tissue were treated with aBL at 216 J/cm2, in vitro, in parallel with unexposed PAO1 or PAO1 isolates from mouse wound infections not treated with aBL. No statistically significant correlation was found between the aBL-susceptibility of bacteria in vitro and the number of cycles of aBL exposure any bacterial species (P ≥ 0.26). In addition, serial exposure of infected mouse wounds to aBL did not result in any change in the susceptibility to aBL of PAO1 (P = 0.97). In conclusion, it is unlikely that sequential exposure to aBL will result in aBL-resistance in Gram-negative bacteria. Also, multiple aBL treatments may potentially be administered to an infected wound without resistance development becoming a concern.